Co-dominant genetic diagnosis test

ABSTRACT

A method for detecting the homozygous or heterozygous state of mutations assumed to be present in a nucleic acid by simultaneously using two primer pairs. The two different primer pairs lead to the production of amplified fragments of different sizes, and the number and quality of the amplified bands enables homozygous and heterozygous items to be distinguished on the basis of said mutation.

The present invention relates to a co-dominant genetic diagnosis test,i.e., a test to distinguish homozygous and heterozygous individuals fora polymorphous allele in a population.

A rapid and effective method for detecting a point mutation at thegenomic DNA level is essential to the identification of polymorphismsboth for genetic studies and predicting a risk of disease linked to thatpolymorphism, and for the study of the molecular bases for hereditarydiseases. It is also essential for the development of a geneticdiagnosis test.

In the remainder of the text, the term “point mutation” is used toencompass a change in a sequence, be it a nucleotide transition,transversion, deletion or addition; more generally, this includestransitions, transversions, deletions or additions of 1 to 6nucleotides.

More generally, such a type of rapid, effective and inexpensivedetection method can be applied to detecting mutations in any livingorganism, be it a micro-organism, animal or plant, the method being ofparticular importance for diploid organsims. Such a type of detection isapplicable to the fields of agriculture and food, medicine or veterinarydiagnoses, or to animal or plant selection.

PCR (1) represented a great advance for genomic DNA analysis. Thattechnique enables genetic diseases to be diagnosed when combined withother techniques (2, 3, 4, 5, 6, 7, 8, 9); it may be a combination ofPCR and direct sequencing (2, 3, 4, 10) or the allele specificoligonucleotide (ASO) technique (11, 12).

In some cases, the appearance of a point mutation can create or destroya recognition site in a restriction enzyme (13); the presence or absenceof the restriction site can be used to cany out diagnoses, as hasrecently been demonstrated for sickle-cell anaemia diagnosis (7); in thesame way, a restriction polymorphism can be linked to a noncharacterised mutation which enables a diagnosis to be made in familiesby analysis of that polymorphism after amplification. A number oftechniques have been developed with the aim of enabling such mutationsto be detected by combining PCR with other types of reaction. These arein particular:

the technique known as PCR amplification of specific alleles (PASA), amodification of the PCR technique using either an oligonucleotide primerwhich hybridises with the wild allele but does not hybridise with themutant allele, or vice versa: the amplified product will thus bespecific for the allele for which the primers have been selected andamplification is thus ineffective if the primer has not hybridised withthe corresponding allele (14);

HEIM et al. (15) used a set of different primers to amplify the twoalleles, the amplifications being followed by allele specific PCR;

SCHUSTER et al. (16) combined asymmetric PCR with allele specific PCRusing a set of 3 oligonucleotide primers in a single reaction mixture todetect a point mutation in the apoB gene; however, for recessivediseases, that (simple) technique cannot distinguish individualscarrying a single mutated allele from diseased individuals with twomutated alleles.

Other methods have been used, particularly for demonstrating thecreation of a restriction site by mutation from an amplificationproduct. That technique has been used to detect haemophilia B (17) orhaemophilia A (18).

None of the diagnosis test techniques described is suitable forwidespread use in a population as they require either lengthy, complex,and expensive steps which necessitate the exercise of a high level oftechnical skill, or they cannot differentiate homozygous fromheterozygous individuals; other techniques such as allele specificoligonucleotides are dominant types and cannot differentiate ahomozygous individual from a heterozygous individual having one normalallele and one mutated allele. For genetic diagnosis, in particularpredicting the risk of genetic disease in given populations, it isextremely important to be able to identify those two populations withoutresorting to complex and expensive techniques.

The present invention can overcome the disadvantages of the differenttechniques described in the literature, and in particular it avoids theuse of radio-elements; it concerns a method of detecting the homoygousor heteroygous state of a mutation assumed to be present in a nucleicacid, characterised in that two nucleic acid amplifications are used,the two amplifications respectively requiring the use of at least twoprimer pairs:

the first pair being constituted by an oligonucleotide which is specificfor the wild allele (A) and a second oligonucleotide (B) which isdifferent from (A);

the second pair being constituted by an oligonucleotide which isspecific for the mutant allele (A′) and a second oligonucleotide (C),which is different from (A′) and from (B);

the difference in length between the amplified fragments between (A) and(B) and between (A′) and (C) being sufficient to be detected byconventional analysis techniques.

These two amplifications are simultaneous.

The term “simultaneous” here means that the reaction products aresimultaneously analysed using conventional methods, in particularanalytical or preparative DNA separation, especially polyacrylamide gelor agarose gel electrophoresis; however, it will be clear to the skilledperson that any other method of analysing the size of the amplifiedfragments (such as chromatography) can be considered as an equivalentmeans in the method of the invention.

The two amplification reactions can be carried out either in twodifferent reaction mixtures if (A) and (A′) are complementary to thesame DNA chain, or they can be carried out in the same reaction mixtureif (A) and (A′) are complementary to the (+) and (−) strands of the DNArespectively.

In particular, the differences in the lengths can be detected by theexistence of two different bands after agarose gel electrophoresismigration, for example; however, it will be clear that when detectionmethods become more sensitive, the differences in the lengths betweenthe amplified fragments can be reduced.

More generally, any technique for amplifying a DNA sequence whichcomprises the use of at least two primers and a polymerase to synthesisethe complementary sequence between the two primers, whatever theimprovements thereto, can be used to implement the invention whichresides in the simultaneous use of two different primer pairs andsimultaneous visualisation of the PCR products.

The two primer pairs, (A) and (B) and (A′) and (C), can be symmetricalor inverted, in other words (A) and (A′) are hybridisable with the samestrand of the double helix, and (B) and (C) with the other strand, or incontrast, the (A) (B) pair and the (A′) (C) pair can be inverted, i.e.,(A) and (A′) are hybridisable with complementary strands of the DNAchain, like (B) and (C).

In the first variation, the two amplifications carried out by the twoprimer pairs must be carried out separately, then the reaction productsmust be mixed for analysis using conventional methods.

In contrast, in the second variation, the reaction products can be mixedfrom the start, since amplification between (A) and (C) cannot occur.However, in the latter case, primers (A) and (A′) must have asufficiently different sequence to avoid unwanted hybridisation between(A) and (A′) in the reaction mixture. The only requirement is that (A)and (A′) carry the nucleotide corresponding to that for which themutation is sought.

Finally, this technique can be used whatever the DNA-containingorganism, be it a micro-organism, bacterium, virus, animal or plant;however it is of particular importance for diploid or polyploidorganisms.

The usefulness of this novel technique has been demonstrated byidentifying a mutation in Amish populations from southern Indianacarrying a gene coding for a protein involved in a recessive autosomaldisease: limb-girdle muscular dystrophy.

While the conventional PCR method has proved to be extremely powerfulfor amplifying target sequences, particularly in complex genomes, smallunwanted bands due to artefacts often appear in the band spectraobtained after amplification. This is often interpreted as a primingerror in the target chain; further, R. H. DON (19) has developed atechnique known as “Touchdown” which can eliminate these artefacts farmore rigorously than prior techniques which were essentially adjustmentsto the concentration of magnesium or an increase in the hybridisationtemperature of the primer with the DNA. The Touchdown method exploitsthe exponential nature of PCR and can begin above the standardhybridisation temperature: the reaction temperature starts 5° C. to 10°C. above this hybridisation temperature (for example 65° C.) thendecreases regularly by 1° C. to 2° C. per cycle until the standardhybridisation temperature is obtained by this technique; any differencein Tm between correct and incorrect annealing gives an advantage of thecorrect product over the incorrect product, everything else being equal.Thus a difference of 5° C. can give a 4⁵ times advantage (19).

In order to avoid the risk of errors inherent to amplificationtechniques, in the method of the invention the amplified fragmentspreferably have lengths which are respectively in the range 50 to 200nucleotides; further, and so that identification of the amplifiedsegments between (A) and (B) and (A′) and (C) is not ambiguous, thedifference in length is preferably at least 10%. The method of theinvention is particularly important for detecting point mutations asdefined above.

The invention also concerns a kit for diagnosing assumed homozygous orheterozygous point mutations and is characterised in that it contains atleast:

a) a heat stable polymerase;

b) a first primer pair constituted by an oligonucleotide (A) which isspecific for the wild allele and a second specific oligonucleotide (B),distinct from (A);

c) a second primer pair constituted by an oligonucleotide which isspecific for the mutant allele (A′) and a second oligonucleotide (C)distinct from (A), the size of the amplified fragments between primers(A) and (B) and primers (A′) and (C) preferably differing by at least10%.

In addition to the above four primers (A), (A′), (B), (C), the kit ofthe invention contains all of the elements required to enable PCRamplification, or any improved method derived therefrom, in particularthe “Touchdown” PCR method, to be carried out.

More particularly, the kit of the invention can be used to detect oridentify the homozygous or heterozygous state of point mutations,whether these mutations are transitions or transversions. Using thesetwo simultaneous amplifications results in a co-dominant diagnosis testthe results of which can be interpreted more easily than any prior artprocedures.

The diagnosis kit of the invention can also be used in the field ofhuman or animal health and in any other sector such as the environment,plant breeding or the agriculture and food industry for which detectionand monitoring of the healthy or infectious state of the medium may beimportant.

The diagnosis test can also be applied to an animal or plant selectionprocess.

The following non limiting examples and the accompanying figuresillustrate the performance of the invention in detecting a pointmutation in the LGMD2E gene coding for the protein involved in a type oflimb-girdle muscular dystrophy.

FIG. 1 is a diagram which illustrates the system of the invention whenthe primer pairs are symmetrical. In this Figure, N is the normal alleleand M is the mutant allele. The arrows point in the 5′-3′ direction andrepresent the primers.

FIG. 2 is a diagram which illustrates the system of the invention whenthe primer pairs are asymmetrical. N and M and the arrows have the samemeanings as in FIG. 1.

FIG. 3 shows the segregation of the threonine to arginine substitutionin an Amish population. In the figure, line A shows the pedigree offamily A620 in which affected or healthy individuals are indicated byblack or white symbols respectively. The term “healthy” means that theindividuals may be non carriers or carriers of heterozygotes; B showsthe result obtained for agarose gel electrophoresis of mixtures of theamplification products using “Touchdown” PCR of fragments of 100 and 158base pairs respectively; C shows the segregation of haplotypes onchromosome 4 in the family; chromosomes carrying the mutation areringed, and CA12T represents the intragenic micro-satellite.

IMPLEMENTATION EXAMPLE

FIGS. 1 and 2 illustrate the technology of the invention. In thisexample, all the primers are 20 nucleotides in length.

a) Primer Pairs (A) (B) and (A′) (C) are Symmetrical

This case is shown in FIG. 1.

In the figure, the first primer pair (A) (B) specific to the normalallele can produce a 180 bp amplification product; primer (B) is located140 bp below the nucleotide where the mutation is located and primer (A)contains that nucleotide at its 3′ extremity.

The second primer pair (A′) (C), which serves to identify the mutantgenotype, has a primer identical to primer (A), but in place of thenormal 3′ nucleotide it has mutant nucleotide M. The other primer (C)below the mutation is selected so that the product obtained by PCR issignificantly different from product (A) (B), in this case 120 bp.

When the amplification products are mixed after the PCR phase thendeposited on 4% agarose gel containing ethidium bromide in aTBE1×buffer, two bands are obtained in the same trace when the startingsample contains a normal allele and a mutant allele. Thus theconventional “dominant” diagnosis system with a “yes/no” type responsefor each of the PCR reactions has been transformed into a readilyinterpretable co-dominant system.

Homoygotes produce only a single band of 120 or 180 bp depending onwhether the homozygote is mutated or normal.

b) Antisymmetrical Primers

FIG. 2 illustrates this case. It can clearly be seen that the primerpairs (A) (B) and (A′) (C) lead, as before, to amplification products of120 bp for the mutant and 180 bp for the wild.

In this figure, it can be seen that when PCR amplification is carriedout in a single reaction medium containing the 4 primers, a mixture of 3amplification products is obtained: 2 products corresponding to the wildand mutant alleles, of respectively 180 bp and 120 bp, and a productcorresponding to amplification between primers (B) and (C) with a lengthof 260 bp.

Depositing the products of the PCR on 4% agarose gel containing ethidiumbromide produces three bands corresponding respectively to the 120, 180and 260 bp products when the amplified DNA is heterozygous and carriesthe two alleles N and M.

If in contrast, the DNA is homozygous for the normal allele, theamplification products will comprise only 2 bands of 180 and 260 bp.

Finally, if the DNA is a mutant homozygote, the reaction product willcontain 2 bands of 120 and 260 bp.

This diagnosis system thus has all of the advantages of other systemsusing PCR, namely sensitivity, non radioactive, automatable, etc. . . ., but above all it has the additional advantage of transforming dominantmarkers into co-dominant markers.

EXAMPLE OF USE Identification of a Mutation in Amish Patients CarryingLGMD2E

a) Selection of Families

An analysis was carried out on six families, already described in theprior art (20), comprising 52 individuals, 13 of whom were affected. 5additional families were also included, comprising 39 individuals intotal, 13 of whom were affected.

When seeking to identify the protein involved in these families,Northern blot analyses were carried out on the total RNA isolated from askeletal muscle biopsy to determine whether the size or quantity ofmessenger RNA of the LGMD2E gene which was produced had been affected.The translated RNA population, with a size of 4.4 Kb was normal inquantity and size both in samples from the affected patients and fromthe healthy controls. This strongly suggested that the mutation wasprobably due to a point mutation as defined above. In order to verifythis, cDNA fragments of the LGMD2E gene were amplified after reversetranscription from total RNA prepared from six muscle biopsies. TheRT-PCR products were sequenced and a simple transversion of C to G atnucleotide 461 was detected in the two patients with two mutatedalleles. The codon change is ACA to AGA and results in a substitution ofthreonine by arginine corresponding to a missense mutation in residue151.

Segregation of this mutation was studied in this family and in otherfamilies carrying limb-girdle muscular dystrophy then amplifying thecorresponding fragment by the Touchdown PCR technique described above.

b) Touchdown PCR

50 ng of DNA underwent the touchdown PCR procedure (19) in 50 μlitres ofreaction mixture containing 10 mM of Tris HCl, pH 8.8, 50 mM KCl, 1.5 mMMgCl₂, 0.1% Triton X-100, 200 mM of each dNTP, 100 ng of each primer,and 2 units of Taq polymerase (Perkin-Elmer). After denaturing at 96° C.for 5 minutes, the first amplification step was carried out as follows:40 seconds (sec) at 94° C. then 30 sec at 63° C. (this constituting acycle) with a reduction of 1° C. every 2 cycles from 63° C. to 59° C. Intotal, 10 PCR cycles were carried out with two cycles at eachtemperature of 63° C. to 59° C. inclusive. The second step was carriedout in 25 additional amplification cycles consisting of 40 sec at 94° C.and 30 sec at 58° C.

Primer Pairs Used

First primer pair:

a) Type AB primer pair to amplify the wild allele:

T461: 5′-GTTTTTCAGCAAGGGACAAC-3′

ml: 5′-CTTTTCACTCCACTTGGCAA-3′.

Second primer pair to amplify the mutant allele:

A461: 5′-GTTTTTCAGCAAGGGACAAG-3′

m3: 5′-TATTTTGAGTCCTCGGGTCA-3′

It should be noted that in T461, the G at the 3′ end has beensubstituted by C at the 3′ end corresponding to a transversion of C to Gin the cDNA sequence.

The amplification products were analysed on 4% agarose gelelectrophoresis stained with ethidium bromide.

c) Results

The results are shown in part B of FIG. 1, the annotation of which hasbeen given above. The difference in the sizes of the amplified segmentswas 58 base pairs, knowing that the amplification products of theT461/ml pair was 100 base pairs and that of the A461/m3 pair was 158base pairs.

Analysis of FIG. 1 shows that parents with a normal phenotype wereheterozygous since the amplification product contained the two types offragments. Clearly, if one of these individuals was normal homozygous,the profile obtained after this group of operations would comprise onlybands corresponding to a molecular weight of 158 base pairs.

Thus for the first time, the technology of the invention canunambiguously distinguish the homozygous or heterozygous state of amutation in a population, avoiding complex enzymatic digestion orrestriction site creation, for example. It is rapid and avoidsexperimentation using radio-elements.

Detection of heterozygotes has grown in importance in the field known aspredictive medicine: for recessive sex-linked diseases, the possibilityof detecting carrier females in families at risk represents aconsiderable advance. Regarding dominant diseases with delayedexpression, carriers of the genetic trait are potential patients andthis type of analysis can detect the risk whatever the penetrance of thedefect and its degree of expressivity. In this case, the method of theinvention enables pre-symptomatic diagnosis to be carried out, i.e.,before the appearance of the first signs of any disease.

Finally, it will be clear to the skilled person that if the disease or,more generally, the phenotype sought, results from a combination ofdifferent point mutations, the principle of amplification using twoprimers and a heat stable polymerase to give amplification products withdifferent sizes for each allele allows for combination of a plurality ofthese primer pairs provided that the amplification products each have adefined size, which is different from one product to another, and arevisualisable.

BIBLIOGRAPHY

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What is claimed is:
 1. A method for detecting the homozygous orheterozygous state of mutations present in a nucleic acid, said methodcomprising the steps of: (1) amplifying two nucleic acids using apolymerase and at least two primer pairs wherein: a first primer pairconsists of an oligonucleotide (A) which is specific for a wild alleleand a second oligonucleotide (B), and a second primer pair consists ofan oligonucleotide (A′) which is specific for a mutant allele and asecond oligonucleotide (C); wherein there is a difference in lengthbetween the amplified fragments between (A) and (B) and between (A′) and(C) respectively; and (2) detecting said nucleic acid fragments.
 2. Themethod according to claim 1, wherein primer (A) of the first pairhybridizes with one DNA strand, and primer (A′) of the second pairhybridizes with the complementary strand.
 3. The method according toclaim 1, wherein primer (A) of the first pair hybridizes with one DNAstrand, and primer (A′) hybridizes with the same DNA strand.
 4. Themethod according to claim 1, wherein amplification is carried out by anamplification method using at least two primers and a polymerase.
 5. Themethod according to claim 1, wherein the mutations are point mutations.6. The method according to claim 1, wherein the nucleic acid in which amutation is detected originates from a human, an animal, or from a humanor animal portion.
 7. The method according to claim 1, wherein thenucleic acid in which the existence of a mutation is detected originatesfrom a plant or a portion thereof.
 8. A diagnosis kit for one stepdetection of homozygous or heterozygous mutations in a nucleic acid,consisting of at least: a) a heat stable polymerase; b) a first primerpair constituted by an oligonucleotide (A) which is specific for thewild allele and a second oligonucleotide (B); c) a second primer pairconstituted by an oligonucleotide (A′) which is specific for the mutantallele and a second oligonucleotide (C); wherein the size of theamplified fragments between primers (A) and (B) and primers (A′) and (C)have a difference which is detectable.
 9. The kit according to claim 8,wherein the size difference between the amplified fragments is at least10%.
 10. The kit according to claim 9, wherein said kit further containselements enabling amplification by PCR or any derivative method.
 11. Thekit according to claim 9, wherein the assumed mutations for diagnosisare point mutations.
 12. The diagnosis kit according to claim 8, fordetecting genetic mutations in man or in animals.
 13. The diagnosis kitaccording to claim 8, for detecting genetic mutations in plants orportions thereof.
 14. The diagnosis kit according to claim 8, for animalor plant selection.
 15. The method according to claim 4, wherein saidamplification method is “Touchdown” PCR.
 16. The method according toclaim 10, wherein said amplification method is “Touchdown” PCR, using atleast two primers.